Abstract
Localization of transcription regulatory proteins in the nucleus is dynamically regulated, and may alter nucleoplasmic concentrations and/or assembly of multimolecular transcription regulatory complexes, which ultimately regulate gene expression. Since growth hormone (GH) regulates multiple transcription factors including C/EBP beta, the effect of GH on the subcellular localization of C/EBP beta was examined in 3T3-F442A preadipocytes. Indirect immunofluorescence shows that C/EBP beta is diffusely distributed in nuclei of quiescent cells. Within 5 min of GH treatment, the diffuse pattern dramatically becomes punctate. The relocalization of C/EBP beta coincides with DAPI staining of heterochromatin. Further, C/EBP beta and heterochromatin protein (HP)-1 alpha colocalize in the nucleus, consistent with localization of C/EBP beta to pericentromeric heterochromatin. In contrast, C/EBP delta exhibits a diffuse distribution in the nucleus that is not modified by GH treatment. C/EBP beta is rapidly and transiently phosphorylated on a conserved MAPK consensus site in response to GH (Piwien-Pilipuk, G., MacDougald, O. A., and Schwartz, J. (2002) J. Biol. Chem. 277, 44557-44565). Indirect immunofluorescence using antibodies specific for C/EBP beta phosphorylated on the conserved MAPK site shows that GH also rapidly induces a punctate pattern of staining for the phosphorylated C/EBP beta. In addition, phosphorylated C/EBP beta colocalizes to pericentromeric heterochromatin. The satellite DNA present in heterochromatin contains multiple C/EBP binding sites. DNA binding analysis shows that C/EBP beta, C/EBP delta, and C/EBP alpha (p42 and p30 forms) can bind to satellite DNA as homo- or heterocomplexes in vitro. Importantly, GH rapidly and transiently increases binding of endogenous C/EBP beta from 3T3-F442A cells to satellite DNA. Further, the GH-promoted nuclear relocalization of C/EBP beta to pericentromeric heterochromatin was prevented by the MEK inhibitor U0126. This observation suggests that GH-dependent MAPK activation plays a role in the regulation of nuclear relocalization of C/EBP beta. Nuclear redistribution introduces a new level of transcriptional regulation in GH action, since GH-mediated phosphorylation and nuclear redistribution of C/EBP beta may be coordinated to achieve spatial-temporal control of gene expression.
Highlights
Chromatin is organized into higher order fibers in interphase chromosomes, which localize into discrete territories, providing an excellent scenario for establishing spatio-temporal regulation of gene transcription [1, 2]
Since CCAAT/enhancer-binding protein (C/EBP) is a ubiquitously distributed transcription factor regulated by growth hormone (GH) by changes in its phosphorylation state (18 –20), the subcellular distribution of C/EBP was investigated in GH-treated cells
Overlay of nuclear images from cells treated with GH shows that C/EBP and DAPI staining do coincide (Fig. 1a, panel J), suggesting that GH promotes the relocalization of C/EBP to areas of heterochromatin
Summary
C/EBP, CCAAT/enhancer-binding protein; DAPI, 4Ј,6-diamidino-2-phenylindole; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; SFC, splicing factor compartment; HP, heterochromatic protein; GH, growth hormone; LAP, liver-enriched-activating protein; LIP, liver-enriched inhibitory protein. C/EBP and ␦, which are diffusely distributed in the nuclei of 3T3-L1 preadipocytes, acquire a punctate pattern of nuclear distribution 12–24 h after induction of differentiation, which corresponds to localization to pericentromeric heterochromatin [22]. C/EBP␣ localizes in areas of pericentromeric heterochromatin concomitant with cessation of mitotic clonal expansion of the cells [22]. These observations suggest that the subnuclear distribution of C/EBPs may be associated with their roles during differentiation. The rapid nuclear relocalization of C/EBP is blocked by MAPK inhibitors, suggesting that MAPK activation is required for GH-induced nuclear redistribution of C/EBP.
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